*3.2. Di*ff*erentially Expressed Genes in K133AS-R vs. K133WT*

β The data for K133AS-R revealed several differentially expressed genes, which are expected to contribute to drug resistance. Extensive variation in the expression of several genes like pteridine transporter and histone-encoding genes was observed in artemisinin-resistant isolates. Marked variation in the number of peptidases, such as metallopeptidase (LDBPK\_330210), aminopeptidase P1 (LDBPK\_020010), and lipases (LDBPK\_341140), was also observed in K133AS-R. Enzymes involved in the lipid biochemical pathway, such as fatty acid elongation and fatty acid desaturation, were affected in artemisinin-resistant *Leishmania*, suggesting a decreased fluidity of the parasite membrane, which may be contributing towards drug resistance as observed in the case of miltefosine-resistant parasite [60]. Genes encoding phosphoglycan β-1,3 galactosyltransferase (involved in glycosylation of proteins), ATP binding cassette transporters (ABC transporters), ABCA2, ABCA7, and ABCA8 exhibited one missense and two frameshift mutations having moderate and high impact in K133AS-R parasites. Additionally, changes in folate/biopterin transporter (upstream gene variant, impact modifier), P-type H+-ATPase (frameshift mutation), and UDP-galactose transporter have been observed in the AS-R parasite. Cell surface protein-encoding genes viz. amastin-like proteins, and proteophosphoglycan (ppg3)-related protein displayed mutation in the K133AS-R isolate. Alterations in ceroidlipofuscinosis neuronal protein 3 (CLN3, LDBPK\_061360) responsible for *Leishmania* virulence were also observed, showing six mutations, including missense mutation with moderate impact, which may have a direct effect on lysosomal function [61]. Moderate impacts on enzymes of the TCA cycle viz. citrate synthase, pyruvate kinase, and succinate dehydrogenase were noted in K133AS-R. In addition, specific genes present in K133AS-R that encode peptidase-like cysteine peptidase B, serine peptidase, heat shock proteins, upstream gene variants, and downstream gene variant with modifier impact are speculated to have direct or indirect role in pathogenesis. Interestingly, two novel gene mutations in K133AS-R including apical membrane antigen1 (AMA1, LDBPK\_301480) (moderate impact, missense mutation) and cathepsin L-like protease were identified, whose role in pathogenesis has been reported previously [62].
